Project Summary The goal of my thesis project is to dissect the direct effects of MYC dysregulation, mediated by different types of genetic lesions found in human cancer, on the regulation of hematopoiesis. Alterations of the MYC locus are commonly found in multiple malignancies in the form of translocations, amplifications and mutations. Most of these genetic alterations are believed to result in dysregulated MYC protein expression. However, amplified and mutated MYC loci typically maintain all local cis-regulatory elements, while translocated MYC loci lose them upon juxtaposition to heterologous regulatory regions and are therefore expected to be unresponsive to external cell arrest signals, in contrast to amplified or mutated loci. Altered MYC expression during developmental processes has been shown to result in abnormal cellular proliferation and differentiation, effects with direct relevance to oncogenesis.! MYC overexpression has also been shown to result in aberrant patterns of DNA binding referred to as ?enhancer invasion?, whereby excess MYC binds to active gene regulatory enhancers. Enhancers are transcriptional regulatory regions critical for the control of development and cell fate transitions and altered enhancer regulation has recently been proposed as a potential oncogenic mechanism. While the functional consequences of MYC binding to active enhancers are unclear, altered enhancer activity could represent a mechanism by which MYC dysregulation translates into biological effects relevant for cancer pathogenesis. MYC expression is tightly regulated in hematopoietic cells, and its overexpression has been shown to disrupt normal hematopoiesis in mice. I hypothesize that the genomic context underlying MYC dysregulation (translocation, amplification or mutation) influences the occurrence of enhancer invasion events, which can in turn alter enhancer function and disrupt cell differentiation during hematopoiesis, contributing to oncogenic transformation. In order to functionally dissect the direct effects of MYC dysregulation driven by different genetic mechanisms on enhancer regulation during human hematopoiesis, I aim to: (1) develop novel models of conditional MYC dysregulation during human Embryonic Stem Cell (hESC)-derived hematopoiesis, and (2) characterize MYC promiscuous enhancer binding and its functional consequences during hematopoietic differentiation. This project aims to analyze for the first time the functional differences between different modes of MYC dysregulation (amplification, translocation, and mutation) by utilizing cutting edge technologies, such as human pluripotent stem cells and CRISPR/Cas9 genome editing, to generate highly innovative models of these 3 forms of MYC dysregulation. Furthermore, this work will characterize for the first time in detail MYC binding at enhancers under different genetic modes of MYC dysregulation, and the biological consequences of this phenomenon during hematopoiesis.